ABSTRACT Cryo-electron microscopy (cryo-EM) is revolutionizing the field of structural biology by providing advantages over long-standing and more frequently used techniques including x-ray crystallography and nuclear magnetic resonance. Recent technological advancements have begun to expand the number and types of proteins that can be characterized using cryo-EM; however, a major barrier to the widespread adoption of the technique still exists. Namely, an inverse correlation exists between the molecular weight of the target protein and the resolution that can be achieved by electron microscopy, thereby limiting the utility of the technique to very large proteins or protein complexes. At present, only proteins larger than ~100,000 Daltons routinely give rise to data with resolutions that rival those obtained using x-ray crystallography. Current approaches to circumvent this problem generally rely on increasing the physical bulk of the target protein, often by identifying proteins that specifically interact with the protein under study. A frequently employed method of achieving this is to evolve highly specific antibodies against the target protein, which are then bound to the target protein in the form of Fabs. While general, this method suffers from the drawbacks that new antibodies must be developed for each target protein, which often requires the use of animals and is time consuming and costly. Furthermore, no control over the site of Fab binding on the target is afforded using this method. Here, we propose to address this challenge by developing a single residue ?epitope? in the form of a non-canonical amino acid (NCAA) that is specifically recognized by an existing antibody. Using the well- established amber stop codon suppression technology, NCAAs can be site-specifically incorporated at essentially any position in a target protein. Antibodies raised against the NCAA would then be expected to specifically bind a target protein in which a surface-exposed residue had been replaced with the NCAA. Because this approach decouples the epitope bound by the antibody from features of the target protein, it obviates the need to evolve a new antibody for each protein under study and also affords direct control over the region of the protein targeted by the Fab. We will begin to explore this possibility in two focused aims. We will first use a previously reported antibody against the drug nicotine to probe variants of the protein ferritin in which nicotine-containing NCAAs have been incorporated. We will use this model system to identify ideal chemical parameters of the nicotine containing NCAA that optimize Fab binding and create a rigid protein-protein interface. In a second aim, we will explore the generality of our approach in proteins other than ferritin and attempt to push the size limits of cryo-EM by applying our technique to very small proteins. We ultimately hope to generate a new toolkit for high resolution structure determination using cryo-EM that both removes existing limitations regarding the use of Fabs and also allows the use of this technique by resource-limited researchers.